Assays for detecting modified compounds

ABSTRACT

Provided are methods and compositions which are useful for separating, isolating, detecting, and quantifying compounds of interest which have been modified chemically, enzymatically or catalytically from other compounds which have not been so modified. The modifications may take the form of functional groups which are gained, lost or retained by the compounds of interest.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.14/296,756 filed Jun. 5, 2014, which is a division of U.S. applicationSer. No. 12/806,950 filed Aug. 24, 2010 (now U.S. Pat. No. 8,778,614),each of which is hereby incorporated by reference in its entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jun. 28, 2016, isnamed ENZ-92-D1-CON-SL.txt and is 963 bytes in size.

FIELD OF THE INVENTION

This invention relates to methods for identifying compounds of interestthat gain, retain or lose functional groups. More specifically, methodsare provided to isolate, detect, identify and quantify compounds thatare modified by the addition or loss of functional groups.

BACKGROUND OF THE INVENTION

Biological compounds are often modified enzymatically or chemically toadd or lose a functional group. These functional groups can alter thebiological function of the compound significantly. An illustration ofone such biochemical modification is the kinase-mediated conversion ofsphingosine to sphingosine-1-phosphate, a cellular metabolite which hasimportant signaling functions.

Several methods have been used to detect products which have beenmodified or converted through enzymatic processes to gain or lose afunctional group. These include the use of radioisotopes, for example³²P and ³⁵S. As an example, the latter compound is used to detectsulfotransferase activity, where ³⁵S-adenosine 3′-phosphate5′-phosphosulfate (PAPS) is utilized to identify the addition of asulfate to hydroxyl or amine moieties on a variety of xenobiotics andendogenous substrates by sulfotransferases (Weinshilboum et al., FASEBJ. 11:3-14, 1997).

Affinity matrices, thin layer and column chromatography, massspectroscopy analysis and gel electrophoresis are also used to separatecompounds having various functional groups from compounds lacking suchgroups. Some affinity schemes rely on the recognition of new functionalgroups or charged groups on the biological compounds as immunologicalepitopes. An example is antibodies that bind to sulfonated epitopes ofsclerostin, as described in U.S. patent application Ser. No. 12/802,447.Immunoprecipitation is also commonly used to detect methylated DNA. See,e.g., Thu et al., J Cell Physiol. 222:522-31 (2010). Additionally,several products are marketed that depend on the creation of newimmunological epitopes for detecting modifications in biomolecules. See,for example, DELFIA®, LANCE® and AlphaScreen® assays (PerkinElmer,Inc.), IMAP® (Molecular Devices, Inc. and IQ assay and LightSpeed™ (QTLBiosystems).

Due to the importance of phosphorylation in numerous biological systems,and in particular signal transduction systems, a number of assays havebeen developed to detect kinase activity using fluorescent signalingmoieties. See, e.g. Coffin et al., Anal. Biochem. 278:206-212 (2000); Liet al., Anal. Biochem. 384:56-67 (2009); Sun et al., Anal. Chem.77:2043-2049 (2005); Kupcho et al., Anal. Biochem. 317:210-217 (2003);U.S. Pat. Nos. 4,565,790; 4,808,541; 5,527,684; 6,251,581; 6,287,774;6,743,640; 6,996,194; 7,122,383; 7,250,517; 7,262,282; 7,432,070;7,445,894; 7,582,461; and 7,632,651; and U.S. Patent Publications2004/0166515; 2005/0202565; 2005/0227294; and 2008/0318255. Several suchkinase assays utilize Förster resonance energy transfer (“FRET”)interactions to identify the phosphorylated compounds. See, e.g., Ohuchiet al., Analyst 125:1905-1907 (2000); Zhang et al., Proc. Natl. Acad.Sci. U.S.A. 98:14997-15002 (2001); Ting et al., Proc. Natl. Acad, Sci.U.S.A. 98:15003-15008; Kurokawa et al., J. Biol. Chem. 276:31305-31310(2001); Violin et al., J. Cell Biol. 161:899-909 (2003); Hofmann et al.,Bioorg. Med. Chem. Lett. 11:3091-3094 (2001); Nagai et al., Nat.Biotech. 18:313-316 (2000); Sato et al. Nat. Biotech. 20:287-294 (2002);Li et al., Anal. Bioanal. Chem. 390:2049-2057 (2008); Uri et al.,Biochim. Biophys. Acta 1804:541-546 (2010); Rinisland et al., Proc.Natl. Acad. Sci. U.S.A. 101:15295-15300 (2004); Rinisland et al., BMCBiotechnology 5:16 (2005); Rinisland et al., Assay Drug Dev. Technol.2:183-92; European Patent Application EP1748079; LanthaScreen™, LifeTechnologies, Carlsbad Calif. Reviews of kinase assay technologies areprovided in Ishida et al., J. Pharmacol. Sci. 103:5-11 (2007); Olive,Expert Rev. Proteomics 1:327-241 (2004); Jia et al., Curr. Drug Discov.Technol. 5:59-69 (2008); Vogel et al., Expert Opin. Drug Discov.3:115-128 (2008); Zaman, Combinatorial Chem. High Throughput Screen.6:313-320 (2003); Ahsen and Bomer Chem. Bio. Chem. 6:481-490 (2005);Schmidt et al., J. Chromatog. B 849:154-162 (2007); and Jia, ExpertOpin. Drug Discov. 3:1461-1474 (2008).

The present invention provides two alternative approaches to thenon-radioactive detection of compounds modified with functional groups.One approach utilizes physicochemical differences between the unmodifiedand modified compounds to separate the two compounds. The other approachuses dyes that comprise an energy transfer pair, where the configurationof the dyes differs between a compound with a functional group and thesame compound without a functional group. In that approach, theconfiguration that comprises a charged functional group, but not theconfiguration with an uncharged moiety, causes an energy transferinteraction between the dyes. While currently available technologies fordetecting compounds modified with functional groups are generallydirected to the detection of only a single functional group (e.g.,phosphate groups), both approaches disclosed herein provide advantagesin that they are rapid, simple, and quantitative, and can be used withvarious types of compounds (e.g., small molecules, lipids and peptides)and many different functional groups.

SUMMARY OF THE INVENTION

This invention provides assays and compositions for separating,identifying, and quantifying compounds having functional groups.

In some embodiments, a method is provided for isolating a compound whichhas gained at least one functional group. The method comprises (a)providing: (i) a compound which can gain a functional group, thecompound comprising at least one non-radioactive signaling moiety; (ii)a chemical, enzymatic or catalytic source of the functional group whichcan be gained by the compound; and (iii) means to separate the compoundwhich has gained the functional group from the compound which has notgained the functional group; (b) forming a mixture comprising thecompound and the source of the functional group, and incubating themixture under conditions suitable for the compound to gain thefunctional group; and (c) separating and thereby isolating any compoundwhich has gained the functional group from any compound which has notgained the functional group.

In other embodiments, a method is provided for isolating a compoundwhich has lost at least one functional group. The method comprises (a)providing: (i) a compound comprising a functional group and at least onenon-radioactive signaling moiety; (ii) a chemical, enzymatic orcatalytic source which can remove the functional group from thecompound; and (iii) means for separating the compound which has lost thefunctional group from the compound which has not lost the functionalgroup; (b) forming a mixture comprising the compound and the chemical,enzymatic or catalytic source and incubating the mixture underconditions suitable for the compound to lose the functional group; and(c) separating and thereby isolating any compound which has lost thefunctional group from any compound which has not lost the functionalgroup.

Also provided is a method for determining the addition of a chargedfunctional group to an uncharged moiety on a compound. The methodcomprises (a) providing: (i) the compound without the functional group,wherein the compound further comprises one member of an energy transferpair; (ii) a second member of the energy transfer pair, having a chargeopposite to the charge of the functional group; and (iii) a chemical,enzymatic or catalytic source of the functional group capable of addingthe functional group to the compound; (b) forming a mixture comprisingthe compound without the functional group, the second member of theenergy transfer pair, and the source of the functional group andincubating the mixture under conditions suitable for the compound to addthe functional group; and (c) detecting a FRET interaction between themembers of the energy transfer pair, wherein the presence of the FRETinteraction is indicative of the addition of the functional group to thecompound.

In additional embodiments, a method is provided for determining theaddition of an uncharged functional group to a charged moiety on acompound. The method comprises (a) providing (i) the compound withoutthe functional group, wherein the compound further comprises one memberof an energy transfer pair; (ii) a second member of the energy transferpair, having a charge opposite to the charge of the moiety; and (iii) achemical, enzymatic or catalytic source of the functional group capableof adding the functional group to the charged moiety; (b) forming amixture of the compound without the functional group and the source ofthe functional group and incubating the mixture under conditionssuitable for the compound to add the functional group to the chargedmoiety; (c) adding the second member of the energy transfer pair to themixture of step (b); and (d) evaluating the energy transfer pair todetermine the degree to which a FRET interaction occurs, wherein theaddition of the functional group to the charged moiety causes areduction in the FRET interaction.

Further provided is a method for determining the removal of a chargedfunctional group from an uncharged moiety on a compound. The methodcomprises (a) providing: (i) the compound with the functional group,wherein the compound further comprises one member of an energy transferpair; (ii) a second member of the energy transfer pair, having a chargeopposite to the charge of the functional group; and (iii) a means toremove the functional group from the uncharged moiety; (b) forming amixture of the compound with the functional group and the means toremove the functional group and incubating the mixture under conditionssuitable for the functional group to be removed from the compound; (c)adding the second member of the energy transfer pair to the mixture ofstep (b); and (d) determining whether a FRET interaction is occurringbetween the members of the energy transfer pair, wherein the absence ofa FRET interaction indicates that the charged functional group has beenremoved from the uncharged moiety by the means to remove the functionalgroup.

Additionally, a method is provided for determining the removal of anuncharged functional group from a charged moiety on a compound. Themethod comprises (a) providing: (i) the compound with the functionalgroup, wherein the compound further comprises one member of an energytransfer pair; (ii) a second member of the energy transfer pair, havinga charge opposite to the charged moiety; and (iii) a means to remove thefunctional group from the charged moiety; (b) forming a mixturecomprising the compound with the functional group, the second member ofthe energy transfer pair, and the means to remove the functional groupfrom the charged moiety and incubating the mixture under conditionssuitable for the functional group to be removed from the compound; (c)determining whether a FRET interaction is occurring between the membersof the energy transfer pair, wherein the presence of a FRET interactionindicates that the uncharged functional group has been removed from thecharged moiety by the means to remove the functional group.

In further embodiments, a reagent is provided for determining thepresence of an enzyme that adds or removes a charged functional group toan uncharged moiety on a substrate. The reagent comprises the substrateto which a first member and a second member of an energy transfer pairare covalently bound at a distance such that a FRET interaction does notoccur unless the charged functional group is present, wherein the secondmember of the energy transfer pair has a charge opposite to the chargeof the functional group, and wherein the presence of the chargedfunctional group to the substrate causes noncovalent binding of thesecond member of the energy transfer pair to the functional group,bringing the first member and the second member of the energy transferpair close enough so that a FRET interaction occurs.

Additionally, a reagent is provided for determining the presence of anenzyme that adds or removes an uncharged functional group to a chargedmoiety on a substrate. The reagent comprises the substrate to which afirst member and a second member of an energy transfer pair arecovalently bound at a distance such that a FRET interaction does notoccur unless the uncharged functional group is absent, wherein thesecond member of the energy transfer pair has a charge opposite to thecharge of the moiety, and wherein the absence of the unchargedfunctional group causes noncovalent binding of the second member of theenergy transfer pair to the charged moiety, bringing the first memberand the second member of the energy transfer pair close enough so that aFRET interaction occurs.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C are illustrations depicting intermolecular andintramolecular binding between a fluorescently labeled compound and afunctional group added in an enzyme reaction to induce a Försterresonance energy transfer (FRET) interaction between an energy transferpair. FIG. 1A illustrates the energy transfer pair in an intramoleculararrangement on the peptide; FIG. 1B shows the energy transfer pair in anintermolecular arrangement; and FIG. 1C shows the use of a chelator onenergy transfer dye F2 and metal ion to cause binding of a functionalgroup to F2 in an intermolecular arrangement.

FIG. 2 is a graph showing the results of a sphingosine kinase 1 assayusing six different dilutions of the enzyme, where no carrier was usedwhen precipitating the product of the reaction.

FIG. 3 is a graph of the peak results from each assay where the resultsare shown in FIG. 2.

FIG. 4 is a graph showing the results of a sphingosine kinase 1 assayusing six different dilutions of the enzyme, where a carrier was usedwhen precipitating the product of the reaction.

FIG. 5 is a graph of the peak results from each assay where the resultsare shown in FIG. 4.

FIG. 6 is a graph showing the results of a sphingosine kinase 1 assaywith and without sphingosine kinase 1 inhibitors.

FIG. 7 is a graph showing the results of an AKT1 assay measuringphosphorylated crosstide peptide using six different dilutions of theenzyme.

FIG. 8 is a graph of the peak results from each assay where the resultsare shown in FIG. 7.

FIG. 9 is a graph showing the results of a precipitation of variousconcentrations of fluoresceinated, sulfated EBJR peptide.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The following terms and phrases shall have the definitions and meaningsset forth below. Unless defined otherwise, all technical and scientificterms used herein have the same meaning as commonly understood to one ofordinary skill in the art.

As used herein, functional groups are chemical moieties which are added,lost or retained by a compound through chemical or biologicalmodification. Functional groups include charged groups and unchargedgroups. Nonlimiting examples of functional groups are phosphate,sulfonate, carboxyl, amine, sulfone, hydroxyl, acetyl, methyl, acyl,glycosyl, sulfate, thiol, amide and nitro.

As used herein, charged groups are functional groups which as chemicalentities are charged and carry a valence which can be positive ornegative at a given pH. Thus, charged groups include monoionic andpolyionic groups or compounds, and more particularly, monocationic,polycationic, monoanionic and polyanionic groups or compounds. Inaccordance with the present invention, charged groups on biologicalmolecules at physiological pH generally include phosphate, sulfonate,carboxyl, amine, sulfone, sulfate, thiol and amide groups. Nonlimitingexamples of uncharged groups (at physiological pH) include methyl andacetyl groups.

As used herein, normalizing reagents are reagents or compounds that,when added to a mixture, serve to promote precipitation orco-precipitation of a compound. An example of a normalizing reagent isan unlabeled compound having a functional group. Such a normalizingreagent is particularly useful where there is only a small quantity ofthe labeled compound. There, the normalizing reagent provides asufficient amount of the compound to promote precipitation. Depending onthe compound to be precipitated, other examples of normalizing reagentsinclude, without limitation, ions (e.g., metal ions) and solvents suchas ethanol, methanol, isopropyl alcohol, dioxane, and combinationsthereof.

As used herein, blocking agents or blockers are compounds, reagents ormaterials that block or mask functional groups, or sites wherefunctional groups may be added, in compounds of interest, so that thefunctional group of interest can be identified. Blockers are usefulwhere there is more than one site on a compound where a functional groupcan be added, such that adding functional groups to multiple sites canconfound the assessment or quantification of a compound or an enzymethat adds the functional group. Blockers are also useful where there aremultiple functional groups on a compound that cause the compound toprecipitate, for example where a method requires that precipitation beused to separate a compound with one particular functional group from acompound without that functional group. Blocking agents can be utilizedto block, for example, amine groups, sulfhydryl groups, aldehyde groups,carboxylate groups or phosphate groups. Blocking agents or blockers maybe reversible or irreversible in nature.

As used herein, the singular forms “a”, “an” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. Additionally, the use of “or” is intended to include“and/or”, unless the context clearly indicates otherwise.

In some embodiments, a method for isolating a compound which has gainedat least one functional group is provided. The method comprises (a)providing: (i) a compound which can gain a functional group, thecompound comprising at least one non-radioactive signaling moiety; (ii)a chemical, enzymatic or catalytic source of the functional group whichcan be gained by the compound; and (iii) means to separate the compoundwhich has gained the functional group from the compound which has notgained the functional group; (b) forming a mixture comprising thecompound and the source of the functional group, and incubating themixture under conditions suitable for the compound to gain thefunctional group; and (c) separating and thereby isolating any compoundwhich has gained the functional group from any compound which has notgained the functional group. The separated compound can be furtherdetected or quantified by detecting or quantifying the non-radioactivesignaling moiety in the separated compound.

The present invention also encompasses the converse of the above method,i.e., a method for isolating a compound which has lost at least onefunctional group. This method comprises (a) providing: (i) a compoundcomprising a functional group and at least one non-radioactive signalingmoiety; (ii) a chemical, enzymatic or catalytic source which can removethe functional group from the compound; and (iii) means for separatingthe compound which has lost the functional group from the compound whichhas not lost the functional group; (b) forming a mixture comprising thecompound and the chemical, enzymatic or catalytic source and incubatingthe mixture under conditions suitable for the compound to lose thefunctional group; and (c) separating and thereby isolating any compoundwhich has lost the functional group from any compound which has not lostthe functional group.

These methods can be used with any compound known in the art that iscapable of acquiring or losing a functional group. In variousembodiments, the compound of interest comprises a macromolecule or asmall biological compound, the latter being a compound naturally presentin biological systems that is less than 2000, 1000 or 500 molecularweight. Nonlimiting examples of macromolecules and small biologicalcompounds include nucleic acids, abasic nucleic acids, peptide nucleicacids, oligopeptides, polypeptides, proteins, sugars, oligosaccharides,polysaccharides, lipids, glycoproteins, glycolipids, proteoglycans, andlipoproteins. More specific examples of compounds contemplated hereinfor use in the present invention include lipids, glycolipids,lipoproteins, apolipoproteins, cytokines, hormones, sphingosines,sphingolipids, and ceram ides, for example glycosylceram ides such asmonosaccharide ceramides (e.g., glucosylceramide, andgalactosylceramide), and disaccharide ceramides (e.g.,lactosylceramide).

In some embodiments, the compound comprises a protein or an oligo- orpolypeptide which is modified in post-translational modification. Asused herein, an oligopeptide is a linear sequence of about 20 or less,about 19 or less, about 18 or less, about 17 or less, or about 16 orless amino acids. A multitude of post-translational modifications ofpeptides and proteins are known in the art. Nonlimiting examples of suchpost-translational modification reactions contemplated for use with thepresent invention include phosphorylation, acetylation, methylation,acylation, glycosylation, GPI anchor addition, hydroxylation, sulfation,disulfide bond formation, deamidation, and nitration.

In other embodiments, the compound comprises a non-biological compound,e.g., a synthetic or environmental compound. Nonlimiting examplesinclude synthetic xenobiotics (e.g., TCDD), drugs, and petroleumproducts.

It is contemplated that these methods are particularly useful where thechemical, enzymatic or catalytic source of the functional groupcomprises an enzyme (e.g., a kinase) and a chemical comprising thefunctional group (e.g., ATP) that is utilized by the enzyme to add thefunctional group to the compound, As such, the present methods provideassays for a multitude of enzymes that add or remove functional groupsfrom compounds including, for example, kinases, phosphatases,sulfatases, sulfotransferases, acetyltransferases, deacetylases,methylases, demethylases, carboxylases, decarboxylases, glycosylases,amidases, deamidases, aminases and deaminases.

In various embodiments, the methods are used to determine whether thereis an enzyme in the sample that can add the functional group to thecompound, e.g., a kinase or sulfotransferase. The methods are alsouseful for quantifying an enzyme activity in a sample. Also, the methodscan be used to determine the presence or activity of inhibitors thatinhibit enzymes that add or remove functional groups (see, e.g., Example1). Alternatively, the methods can be used to determine the presence orquantity of a source of the functional group, e.g., ATP. In stillanother aspect, the methods are useful for detecting and identifyinginhibitors to enzymes that add or remove functional groups.

In carrying out the present invention, it may be important tospecifically differentiate between different isoforms of an enzyme, forexample two kinase isoforms, e.g., sphingosine kinase 1 (SphK1) andsphingosine kinase 2 (SphK2). When using purified enzymes, this isusually not a problem. However, when carrying out assays with cellextracts or live cells, the simultaneous presence of more than oneisoform can lead to measurements that are the sum of each individualisoform activity. Numerous solutions can be applied to obviate thisproblem in the course of running an assay under these conditions, Forinstance, there are differences in substrate specificity, for example,where SphK1 and SphK2 are differentiated by their ability of usingenatomeric forms of sphingosine as a substrate where Sphkl can use onlythe natural D-erythro form whereas SphK2 can use the L-threo form aswell (Liu et al., Prog. Nucleic Acid Res. Mol. Biol. 71:493-511, 2002).Also, conditions can be adjusted that differentially affect eachenzyme's activity, e.g., high salt inhibits SphK1 and stimulates SphK2,whereas Triton X-100 stimulates SphK1 and inhibits SphK2 (Liu et al 2002op cit.). Lastly, inhibitors may be available that have specificity forone form of an enzyme versus the other. Thus, inhibitors that areselective for SphK2, such as SG14, have been described by Kim et al.,Bioorganic Chemistry & Medicinal Chemistry 13:3475-3485 (2005) and anSphK1 specific inhibitor has been described in US Patent Publication No.2010/0035959 A1 and Paugh et al., Blood 15:1382-1391 (2008). An assaymay thus be carried out where no inhibitor is added to give the totalamount of enzyme activity and each of the isoform specific inhibitorscan be added to give the amount of activity contributed by the unblockedisoform. Elimination of one activity or another to separately test aselected isoforms may also be carried out by the use of null mutants(Allende et al., JBC 279:52487-52492, 2004; Kharel et al., JBC280:36865-36872, 2005; Zemann et al., Blood 107:1454-1458, 2006; Michaudet al., FEBS Letters 580:4607-4612, 2006) or by transfection with siRNAdirected toward one isoform (Klawitter et al., Br J Pharmacol150:271-280, 2007; Lai et al., J Immun 183:2097-2103, 2009).

It is also contemplated by the present invention that isoforms may bedistinguished by using different substrates. For example, assays withsphingosine kinase may also be carried out with sphinganine and/orphytosphingosine substrates since both are recognized by SphK2 (Liu 2002op. cit.).

These methods can be utilized with any sample. The sample utilized forthese methods can be derived or taken from any source, including anybiological or non-biological source, such as clinical samples, forexample, blood urine, feces, saliva, pus, semen, serum, other tissuesamples, fermentation broths, culture media, and environmental sources,for example, plant material, soil and the like. If necessary, theanalyte may be pre-extracted or purified by known methods to concentrateor isolate biological components, or eliminate interfering substances,Non-biological sources include, for example, environmental sources andindustrial sources such as ground water, oil spill sites, industrialeffluent, waste treatment sites, etc.

The separation of the compound with the functional group from thecompound without the functional group can utilize any method. Examplesinclude precipitation, capture, phase separation, chromatography,electrophoresis, and the like, all of which are known in the art.

In some embodiments, the separating means comprises differentialprecipitation. A precipitation reaction is a chemical reaction in whicha solid forms from solution. The solid is called the precipitate. Aprecipitate forms if any possible combination of ions in a solutionforms a salt that is insoluble in water. That is, a precipitate is asalt that is no longer solvated by water.

It is understood that, for optimum precipitation of the compound havingthe charged functional group while minimizing precipitation of thecompound without the charged functional group, the proper solutionconditions (e.g., buffer salt, pH and molarity) must be utilized. Suchconditions are known in the art or can be determined without undueexperimentation for any particular compound.

A number of ions are known in the art as being capable of precipitatingor co-precipitating a compound comprising a charged functional group,including, for example, any of the metals from Group IIIB, Group WB orthe Lanthanide Series, and combinations thereof. The metals of Group IUBinclude scandium (Sc), yttrium (Y), lanthanum (La) and actinium (Ac).The metals of Group IVB include titanium (Ti), zirconium (Zr), hafnium(Hf) and rutherfordium (Rf). Among the metals of the Lanthanide Seriesare cerium (Ce), praseodymium (Pr), neodymium (Nd), promethium (Pm),samarium (Sm), europium (Eu), gadolinium (Gd), terbium (Tb), dysprosium(Dy), holmium (Ho), erbium (Er), thulium (Th), ytterbium (Yb) andlutetium (Lu), In some embodiments, for example where the compoundcomprises a phosphate functional group, lanthanide and zirconium areparticularly useful. Barium is also useful for precipitation ofphosphate containing compounds. See, e.g., Example 1.

In some embodiments, optionally after washing, the label is detected inthe precipitate. In other embodiments, optionally after washing, theprecipitate is solubilized and the label is detected in solution,Resolubilization schemes for many precipitates are known in the art. Forexample, a barium precipitate of a phosphate-containing compound may besolubilized with EDTA, since EDTA has a higher affinity for barium thanphosphate. See, e.g., Example 1.

As an alternative to precipitation, a compound having a functional groupcan be separated from the compound without the functional group bydifferentially capturing the compound with the functional group on amatrix that binds the functional group. Techniques and formats forcapturing analytes and probe-containing materials are known. See, e.g.,U.S. Pat. Nos. 6,221,581 and 7,064,197; U.S. Patent Publication No.20100160182; Loomans et al., Assay and Drug Develop Tech 1:445-453(2003); Barnouin et al., Proteomics 5: 4376-4388 (2005); and Lee et al.,Prostaglandins & other Lipid Mediators 84:154-162 (2007). In theseembodiments, once captured, the un-bound compounds can be washed awayand signal from the captured moiety can be directly observed byfluorescence detection.

Retention on a matrix may involve nonspecific background material inwhich unmodified substrate may bind to the matrix and elicit a falsesignal. In using fluorescent labels or markers on a matrix, suchfluorescent components should be minimized so that they do not quenchthe fluorescent signal.

As an illustration of capture techniques, methods and techniques areknown for capturing free-amine functionalities in proteins or peptidesthat are based on plates (support surfaces), resins or gels. Among thevarious methods and techniques known in the art for capturing aminegroups in macromolecules are maleic anhydride-activated plates,aldehyde-activated agarose beads, CDI-activated agarose, AccQ-Tag Ultracolumns, and CM-Sephadex and CM-Sepharose columns. Matricies that bindvarious other functional groups are also known. For example, compoundswith phosphate functional groups can be isolated on a matrix comprisinga metal ion, for example Fe⁺³, Ga⁺², Zn⁺³, Zn⁺², or Mn⁺². See, e.g.,Kinoshita et al., Mol. Cell. Proteomics 5:749-757 (2006).

As briefly discussed above, a blocker is often useful for blocking atleast one moiety on the compound that interferes with the compoundgaining the functional group, e.g., where there are multiple sites wherethe functional group can attach, and/or a moiety that interferes withthe separation step, e.g., where there are moieties that allowprecipitation or capture of a compound without the functional group towhich the precipitation or capture is to be directed.

Blockers are known that can block many specific functional groups incompounds and macromolecules including, for example, amine groups,sulfhydryl groups, aldehyde groups, carboxylate groups and phosphategroups. Blocking agents or blockers may be reversible or irreversible innature, meaning that their effects can be reversed in some instances tounblock or unmask the groups or sites of blocking, or their effects arenot reversible in other instances so that the blocked or masked groupsor sites cannot be unblocked or unmasked easily or practically.

Nonlimiting examples of blocking agents or blockers for specificfunctional groups include the following:

1. Blocking Amine Groups (NH₂)

-   Irreversible: Sulfo-NHS acetate; Acetic anhydride-   Reversible: Citraconic anhydride; Maleic anhydride

2. Blocking Sulfhydril Groups (SH)

-   Irreversible: N-ethylmaleimide; lodoacetamide-   Reversible: Sodium tetrathionate; Ellman's reagent    [5,5′-dithio-bis-(2-nitrobenzoic acid) or DTNB]; Dipyridyl disulfide

3. Blocking Aldehyde Groups

-   lrrevesible: Reductive amination with Tris or ethanolamine-   Revesible: Imine formation

4. Blocking Carboxylate Groups

-   Esters or amides using carbodiimide activation

5. Blocking Phosphate Groups

-   Reversible: Use of 2-cyanoethyl-   Commonly used blocking groups for hydroxyl functionality include the    following:

6. Blocking Ethers

-   -   Methyl ether. In practice, this group is generally irreversible        since it requires harsh conditions for deprotection.    -   Tetrahydrofuranyl ether. This group can be cleaved under mild        acidic conditions.    -   Trimethylsilyl ether. This group can be cleaved by fluoride ion        easily.    -   t-Butyldimethylsilyl ether. This group can be cleaved by        fluoride ion easily.

7. Blocking Esters

-   -   formate, acetate, chloroacetate, trichloroacetate,        Trifluoroacetate.

8. Blocking Carbonates

-   -   alkyl methyl carbonate, methoxy methyl carbonate, alkyl allyl        carbonate, alkyl p-nitrophenyl carbonate.

9. Blocking Sulfonates

-   -   allyl sulfonate, benzylsulfonate, tosylate, methanesulfonate.

Non-radioactive detection can be carried out using labels, signalingmoieties and methods known in the art and appropriate for the signalingmoiety utilized in the assay. These include direct and indirectsignaling methods. Direct signaling methods and labels can utilize, forexample, fluorescent compounds, phosphorescent compounds,chemiluminescent compounds, chromogenic compounds, chelating compounds,electron dense compounds, magnetic compounds, energy transfer compoundsor intercalating compounds, or a combination of any of the foregoing.Indirect signal methods and labels can utilize, for example, antibodies,antigens, haptens, receptors, hormones, ligands or enzymes, or acombination of any of the foregoing.

As is known in the art, the methods can use binding partners inconnection with non-radioactive detection. To this end, such bindingpartners can include, for example, pairs comprising sugars/lectins,antigens/antibodies, ligand/receptor, hormone/receptor,enzyme/substrate, biotin/avidin, biotin/streptavidin, haptens/antibodies(e.g., digoxygenin/anti-digoxygenin), or combinations of any of theforegoing.

Examples of fluorescent dyes useful for the instant methods includexanthene dyes, rhodamine dyes, fluorescein dyes, cyanine dyes,asymmetric cyanine dyes, phthalocyanine dyes, squarene dyes, acridonedyes, quinacridone dyes, bodipy dyes, nitrobenzoxadiazole dyes andfluorescent proteins. When more than one fluorescent label is used,e.g., if performing the methods to detect enzymes for more than onefunctional group, or if combining the method with another assay thatutilizes fluorescent dyes, it is important to choose fluorescentmoieties with different extinction coefficients and spectracharacteristics.

These methods can be carried out in solution as a one step homogeneousassay or as a two step assay involving a matrix or solid support inwhich one or more washing steps are used.

In various embodiments of the present invention, the methods are carriedout in a homogenous format. In other assays, the means to separate thefunctionalized compound from the non-functionalized compound utilizes amatrix that binds the functional group, for example a matrix comprisinga dinuclear metal (e.g., Zn⁺² or Mn⁺²) to bind a phosphate functionalgroup (Kinoshita et al., Mol. Cell. Proteomics 5:749-757, 2006).Detecting the label on the labeled compound bound to the matrix can becarried out either directly or indirectly.

In performing the assay either on a solid matrix or in solution, tubesor microtiter plates can be used to hold the mixture containing thevarious components.

Kits may be designed and assembled for the purpose of carrying out thepresent invention where a non-radioactively labeled kinase substrate isprovided. Reagents for performing the precipitation step may also beprovided in such kits or one or more of these they may be left to theuser to provide.

One embodiment of these methods is described in Example 1, describing anassay for sphingosine kinase comprising combining fluoresceinatedsphingosine, ATP and the kinase to form fluoresceinatedsphingosine-1-phosphate, which was then quantitatively precipitated withbarium acetate and ethanol. In some cases, a normalizing reagent in theform of unlabeled sphingosine-1-phosphate was added. After washing, theprecipitated reaction product was resolubilized and fluorescence wasmeasured. Quantitative precipitation of the phosphorylated reactionproduct was noted, both with and without the normalizing reagent.

Another embodiment of these methods is described in Example 2,describing an assay for the kinase AKT1 comprising combining thefluoresceinated 11-mer oligopeptide crosstide, ATP and AKT1 to formfluoresceinated crosstide phosphate, which was then quantitativelyprecipitated with lanthanum. A normalizing reagent (unfluoresceinatedcrosstide phosphate) was used. After washing, the precipitated reactionproduct was resolubilized and fluorescence was measured. Quantitativeprecipitation of the phosphorylated reaction product was noted.

Still another embodiment of these methods is described in Example 3,where precipitation of a fluoresceinated, sulfated peptide isdemonstrated, showing that a compound with a monovalent anion (sulfate)can be selectively precipitated away from the unmodified peptide.

The three examples provided establish that the instant methods can beutilized to detect and quantify any small molecule or macromoleculehaving a charged functional group.

In some embodiments, non-radioactive signaling moieties are utilizedthat form energy transfer pairs, where the signal is influenced byFörster resonance energy transfer (also known as fluorescence resonanceenergy transfer, or FRET). FRET uses two fluorophores (an energytransfer pair) where the emission spectrum of one fluorophore (thedonor) is of higher energy (having a shorter wavelength) and overlapsthe absorption spectrum of the other fluorophore (the acceptor). Whenthe two fluorophores are brought within about 10-100 Å and the donorfluorophore is excited, the energy of the donor is transferred to theacceptor by a resonance induced dipole-dipole interaction. Thisinteraction is observed by fluorescence quenching of the donorfluorophore and/or emission of the acceptor fluorophore. See, e.g.,discussion in U.S. Pat. No. 6,117,635 and references cited therein.

The FRET interaction forms the basis of additional methods of thisinvention. In these methods, a non-radioactive signaling moietycomprises the two members of an energy transfer pair. At least one ofthe two members of the energy transfer pair is on a compound, within10-100 Å of a site where a functional group (e.g., a phosphate) can beattached, e.g., by a kinase. The second member of the energy transferpair has a charge opposite to the functional group (if the functionalgroup is charged, e.g., a phosphate or sulfate) or to the moiety that aneutral functional group is bound (e.g., where a methyl or acetyl groupis bound to an amino moiety). A FRET interaction thus occurs where thesecond member of the energy transfer pair binds by charge interaction tothe charged functional group or moiety. This can be observed by aquenching of the fluorescence of the donor or an excitation of theacceptor. This forms the basis for methods of determining (1) theaddition of a charged functional group to an uncharged moiety on acompound, (2) the removal of a charged functional group from anuncharged moiety on a compound, (3) the addition of an unchargedfunctional group to a charged moiety on a compound, and (4) the removalof an uncharged functional group from a charged moiety on a compound.Details of these methods are as follows.

In some embodiments, the present invention provides a method fordetermining the addition of a charged functional group to an unchargedmoiety on a compound. The method comprises (a) providing: (i) thecompound without the functional group, wherein the compound furthercomprises one member of an energy transfer pair; (ii) a second member ofthe energy transfer pair, having a charge opposite to the charge of thefunctional group; and (iii) a chemical, enzymatic or catalytic source ofthe functional group capable of adding the functional group to thecompound; (b) forming a mixture comprising the compound without thefunctional group, the second member of the energy transfer pair, and thesource of the functional group and incubating the mixture underconditions suitable for the compound to add the functional group; and(c) detecting a FRET interaction between the members of the energytransfer pair, wherein the presence of the FRET interaction isindicative of the addition of the functional group to the compound.

In other embodiments, a method of determining the addition of anuncharged functional group to a charged moiety on a compound isprovided. The method comprises (a) providing (i) the compound withoutthe functional group, wherein the compound further comprises one memberof an energy transfer pair; (ii) a second member of the energy transferpair, having a charge opposite to the charge of the moiety; and (iii) achemical, enzymatic or catalytic source of the functional group capableof adding the functional group to the charged moiety; (b) forming amixture of the compound without the functional group and the source ofthe functional group and incubating the mixture under conditionssuitable for the compound to add the functional group to the chargedmoiety; (c) adding the second member of the energy transfer pair to themixture of step (b); and (d) evaluating the energy transfer pair todetermine the degree to which a FRET interaction occurs, wherein theaddition of the functional group to the charged moiety causes areduction in the FRET interaction.

Further provided is a method for determining the removal of a chargedfunctional group from an uncharged moiety on a compound of interest. Themethod comprises (a) providing: (i) the compound of interest with thefunctional group, wherein the compound further comprises one member ofan energy transfer pair; (ii) a second member of the energy transferpair, having a charge opposite to the charge of the functional group;and (iii) a means to remove the functional group from the unchargedmoiety; (b) forming a mixture of the compound of interest with thefunctional group and the means to remove the functional group andincubating the mixture under conditions suitable for the functionalgroup to be removed from the compound; (c) adding the second member ofthe energy transfer pair to the mixture of step (b); and (d) determiningwhether a FRET interaction is occurring between the members of theenergy transfer pair, wherein the absence of a FRET interactionindicates that the charged functional group has been removed from theuncharged moiety by the means to remove the functional group.

Additionally, a method for determining the removal of an unchargedfunctional group from a charged moiety on a compound is provided. Themethod comprises (a) providing: (i) the compound with the functionalgroup, wherein the compound further comprises one member of an energytransfer pair; (ii) a second member of the energy transfer pair, havinga charge opposite to the charged moiety; and (iii) a means to remove thefunctional group from the charged moiety; (b) forming a mixturecomprising the compound with the functional group, the second member ofthe energy transfer pair, and the means to remove the functional groupfrom the charged moiety and incubating the mixture under conditionssuitable for the functional group to be removed from the compound; (c)determining whether a FRET interaction is occurring between the membersof the energy transfer pair, wherein the presence of a FRET interactionindicates that the uncharged functional group has been removed from thecharged moiety by the means to remove the functional group.

The detection of a compound with a charged functional group by thesemethods is illustrated in FIGS. 1A-1C. In these embodiments, the assaycan use an intramolecular interaction (FIG. 1A) or an intermolecularinteraction (FIG. 1B). It is noted that an intermolecular interactioncan occur in the configuration illustrated in FIG. 1A, since the F2group on one molecule can bind to the functional group on anothermolecule. When the functional group is not added to the molecule ofinterest, as depicted in (a), energy transfer pair members F1 and F2 aretoo far apart to undergo a FRET interaction. Upon addition of thecharged functional group (denoted as a half circle), as depicted in (b),a moiety of the opposite charge that is on F2 (denoted as an open stemand cup-like structure) binds noncovalently to the functional group bycharge interaction, as depicted in (c), bringing F1 and F2 close enoughto cause a FRET interaction. As discussed above, this FRET interactioncan be detected upon excitation of the energy donor (which can be eitherF1 or F2) by quenching of emittance (e.g., fluorescence) from the energydonor due to energy transfer to the acceptor, or by emittance (e.g.,fluorescence) of the acceptor.

In these embodiments, the charge on the moiety on F2 is of oppositepolarity as the charge of the added functional group, in order for themoiety to bind to the functional group, bringing F1 and F2 close enoughto allow a FRET interaction. Thus, where a negatively charged functionalgroup is added, for example a phosphate, carboxyl, sulfate, nitro, oracyl group, a positively charged dye (F2) is utilized. Conversely, wherea positively charged functional group is added, e.g., where an aminogroup is added in an amination reaction, a negatively charged dye (F2)is used. A majority of fluorescent energy transfer dyes are negativelycharged, with other fluorescent energy transfer dyes, such assulfonamide dyes, being positively charged. Among such positivelycharged sulfonamide dyes are those disclosed in U.S. Pat. Nos. 7,569,695and 7,737,281, Still other dyes are uncharged.

In some embodiments, the added functional group is uncharged, forexample in a methylation or an acetylation reaction. Where the unchargedgroup is added to a charged moiety (e.g., an amino group), thosefunctionalization reactions can be detected using the same formats asillustrated in FIGS. 1A-1C, except the functionalization reactionresults in a lack of a FRET interaction, and the control reaction, wherethe charged moiety is unmodified, would be detected as a FRETinteraction. Thus, the methylation or acetylation reaction is detectedby an increase in the energy donor emittance (e.g., fluorescence) (dueto cessation of quenching) or a decrease in the energy acceptoremittance (e.g., fluorescence) (due to a cessation of energy transferfrom the donor).

In other embodiments, a FRET interaction is utilized to detect theremoval of a functional group from a compound of interest. The methodsand compositions depicted in FIGS. 1A-1C are also used here. Where acharged group is removed to leave an uncharged moiety on the compound ofinterest, for example with the action of a phosphatase, a sulfatase, adeaminase, a decarboxylase, a deamidase or a deaminase, the assay worksin the reverse direction from that depicted in FIGS. 1A-1C. Before theremoval of the functional group, the reagents of FIGS. 1A-1C have theconfiguration of (c), due to the interaction of the charged group withF2. This is detected by a FRET interaction, where the energy donoremittance (e.g., fluorescence) is quenched. With removal of thefunctional group, the FIGS. 1A-1C reagents are as in (a) since theneutral moiety where the functional group was removed does not interactwith the charged F2 dye. Thus, the action of an enzyme that removes acharged functional group to leave a neutral moiety on the compound ofinterest is detected by the elimination of a FRET interaction.

Conversely, the removal of an uncharged group to leave a charged moietyon the compound of interest, for example the removal of a methyl oracetyl group from an amino moiety (e.g., an c amino group of a lysineresidue on a protein) by a demethylase or deacetylase, can be detectedby utilizing the reagent depicted in (a) of FIG. 1A or 1B. The compoundof interest with the uncharged functional group, prepared as a FIGS.1A-1C reagent, has the configuration of (a), since the charged F2 dyedoes not interact with the uncharged functional group. As discussedabove, there is no FRET interaction in the step (a) configuration. Uponremoval of the uncharged functional group, leaving a charged moiety, thereagent takes on the configuration of (c), where a FRET interactiontakes place, due to the interaction of the charged moiety with theoppositely charged F2 dye. Thus, the removal of the functional group isdetected by the presence of the FRET interaction, e.g., the quenching ofthe energy donor of the FRET pair.

For any of the above-described methods, where the F2 dye is notcovalently joined to the compound, as depicted in FIG. 1B, the F2 dyecan be added before the mixture is incubated or after the mixture isincubated. The latter option is useful if the preferred incubationconditions (e.g., temperature, pH, molarity, buffering capacity) topromote enzyme action to add or remove the functional group aredifferent from the preferred conditions under which the F2 dye binds andthe FRET interaction occurs. An important consideration here is that thepH under which the F2 dye binds must be such that the F2 dye and thefunctional group or moiety are charged or uncharged as needed. Suchincubation conditions can be determined for any particular enzyme,compound, and electron transfer pair without undue experimentation.

When designing the particular configurations for these methods, careshould be taken to minimize interference to the method's properexecution. Such interference can come from several sources. In the firstinstance, interference could come from the source of the functionalgroup (e.g., ATP for a kinase reaction). Since in most cases that sourcehas the same charge as the functional group on the compound (forexample, ATP is negatively charged, as is a phosphate functional grouptransferred from ATP to the compound by a kinase), that source competeswith the functional group transferred to the compound for binding to theF2 dye, Such interference can be addressed by means known in the art,for example by minimizing the concentration of the source, and/or byutilizing the intermolecular configuration as depicted in FIG. 1B,and/or by adjusting the conditions after incubation to minimize theeffect of the source interference, e.g., by adjusting the pH or molarityof the solution, and/or by separating the compound from the source (forexample by ultrafiltration or precipitation).

Depending on the structure of the compound, another source ofinterference can be from charged groups on the compound, which couldbind nonspecifically to the F2 dye. This interference can be avoided byuse of a blocker to block the charged groups on the compound, and/or byutilizing an intramolecular configuration as depicted in FIG. 1A, suchthat the F2 moiety is sterically prevented, for example with a linkermoiety, from interacting with charged moieties other than the desiredfunctional group.

An additional source of interference can come from nonspecificinteractions (i.e., stickiness) between the F1 fluorophore and the F2fluorophore, where F1 and F2 are either on the same compound or ondifferent compounds. Such nonspecific interactions can be resolved bythe use of an F1 dye that has the same charge as the F2 dye, or by usingan F1 dye with a neutral charge. When the F1 dye has the same charge asthe F2 dye, care should be taken to minimize interaction of the F1 dyewith the functional group on the same compound or on a differentcompound.

In some embodiments, the fluorescent dye that interacts with thefunctional group (F2 in FIGS. 1A-1C) further comprises a moiety thatcovalently or noncovalently binds to the functional group. An example isa metal chelator which, in the presence of a metal ion that binds to thefunctional group, will promote the binding of the functional group tothe fluorescent dye. This is illustrated in FIG. 1C, where the enzymaticreaction adds a functional group (e,g., phosphate) that can bind metalion (M) to the compound. With the addition of the metal ion and F2comprising a chelator (illustrated as two arms with black knobs), themetal ion binds to the chelator and the functional group, bringing theF2 dye to the proximity of the F1 dye, allowing a FRET interaction. Anonlimiting example of such chelators is the lanthanum (a phosphatebinding metal) chelators described in U.S. Patent No, 5,656,433.Although FIG. 1C shows the metal ion binding to the functional groupbefore binding to the chelator-F2 complex, the opposite could occur—thechelator-F2 complex could be first mixed with the metal ion, allowingthe metal ion to bind to the complex first, and this could then be addedto the enzyme reaction mix.

As with the previously described methods, these methods can be used withany compound known in the art that is capable of acquiring or losing afunctional group. In various embodiments, the compound of interestcomprises a macromolecule or a small biological compound, the latterbeing a compound naturally present in biological systems that are lessthan 2,000, 1,000 or 500 molecular weight. Nonlimiting examples ofmacromolecules and small biological compounds include nucleic acids,abasic nucleic acids, peptide nucleic acids, oligopeptides,polypeptides, proteins, sugars, oligosaccharides, polysaccharides,lipids, glycoproteins, glycolipids, proteoglycans, and lipoproteins.

In some embodiments, the compound comprises a protein or an oligo- orpolypeptide which is modified in post-translational modification. Inother embodiments, the compound comprises a non-biological compound,e.g., a synthetic or environmental compound.

It is contemplated that these methods are particularly useful where thesource of the functional group comprises an enzyme (e.g., a kinase) anda chemical comprising the functional group (e.g., ATP). As such, thesemethods provide assays for a multitude of enzymes that add or removefunctional groups from compounds including, for example, kinases,phosphatases, sulfatases, sulfotransferases, acetyltransferases,deacetylases, methylases, demethylases, carboxylases, decarboxylases,glycosylases, amidases, deamidases, aminases and deaminases.

In various embodiments, the methods are used to determine whether thereis an enzyme in the sample that can add the functional group to thecompound, e.g., a kinase, phosphatase, or sulfotransferase. The methodsare also useful for quantifying an active enzyme in a sample. Also, themethods can be used to determine the presence or activity of inhibitorsthat inhibit enzymes that add or remove functional groups (see, e.g.,Example 1). Alternatively, the methods can be used to determine thepresence or quantity of a source of the functional group, e.g., ATP. Instill another aspect, the methods are useful for detecting and identifyinhibitors to enzymes that add or remove functional groups.

Also provided are reagents for determining the presence of an enzymethat adds or removes a charged functional group to an uncharged moietyon a substrate. The reagents comprise the substrate to which a firstmember and a second member of an energy transfer pair are covalentlybound at a distance such that a FRET interaction does not occur unlessthe charged functional group is present, wherein the second member ofthe energy transfer pair has a charge opposite to the charge of thefunctional group, and wherein the presence of the charged functionalgroup to the substrate causes noncovalent binding of the second memberof the energy transfer pair to the functional group, bringing the firstmember and the second member of the energy transfer pair close enough sothat a FRET interaction occurs.

These reagents are particularly useful for the relevant methodsdescribed above that utilize a FRET interaction. As such, the scope ofthese reagents is contemplated to be the full scope that is useful forany of the above-described methods that utilize FRET and a chargedfunctional group. Thus, the substrate can be any substrate for anyenzyme that adds or removes a charged functional group to an unchargedmoiety. In some embodiments, the substrate is a macromolecule or a smallbiological compound, for example a protein or an oligo- or polypeptidewhich is modified in post-translational modification. In otherembodiments, the compound comprises a non-biological compound, e.g., asynthetic or environmental compound. Nonlimiting examples of enzymesthat catalyze the addition or removal of the charged functional group tothe substrate include kinases, phosphatases, sulfatases,sulfotransferases, carboxylases, decarboxylases, glycosylases, amidases,deamidases, aminases and deaminases.

In various embodiments, the second member of the energy transfer pairfurther comprises a chelator that chelates a metal that binds to thefunctional group, as discussed above.

Additionally, a reagent for determining the presence of an enzyme thatadds or removes an uncharged functional group to a charged moiety on asubstrate is provided. The reagent comprises the substrate to which afirst member and a second member of an energy transfer pair arecovalently bound at a distance such that a FRET interaction does notoccur unless the uncharged functional group is absent, wherein thesecond member of the energy transfer pair has a charge opposite to thecharge of the moiety, and wherein the absence of the unchargedfunctional group causes noncovalent binding of the second member of theenergy transfer pair to the charged moiety, bringing the first memberand the second member of the energy transfer pair close enough so that aFRET interaction occurs.

These reagents are also useful for the relevant methods described abovethat utilize a FRET interaction. As such, the scope of these reagents iscontemplated to be the full scope that is useful for any of theabove-described methods that utilize FRET and an uncharged functionalgroup. Thus, the substrate can be any substrate for any enzyme that addsor removes an uncharged functional group to a charged moiety. In someembodiments, the substrate is a macromolecule or a small biologicalcompound, for example a protein or an oligo- or polypeptide which ismodified in post-translational modification. In other embodiments, thecompound comprises a non-biological compound, e.g., a synthetic orenvironmental compound. Nonlimiting examples of enzymes that catalyzethe addition or removal of the uncharged functional group to thesubstrate include acetyltransferases, deacetylases, methylases anddemethylases.

Preferred embodiments are described in the following examples. Otherembodiments within the scope of the claims herein will be apparent tothose skilled in the art from consideration of the specification orpractice of the invention as disclosed herein. It is intended that thespecification, together with the examples, be considered exemplary only,with the scope and spirit of the invention being indicated by theclaims, which follow the examples.

EXAMPLE 1 Detection of Sphingosine-1-Phosphate Introduction

An assay is described herein where the compound sphingosine-1-phosphate,labeled with a fluorescent dye, is separated from dye-labeledsphingosine by precipitating the phosphorylated compound underconditions where the unphosphorylated compound is not precipitated. Theprecipitate is separated from the unprecipitated sphingosine, thensolubilized, and the dye is quantified, thus quantifying thephosphorylated compound. This assay is particularly useful for detectingand quantifying sphingosine kinase activity.

Sphingosine is 18-carbon amino alcohol with an unsaturated hydrocarbonchain having the structure

Sphingosine is phosphorylated at the non-polar end by the enzymessphingosine kinase 1 and sphingosine kinase 2, to formsphingosine-l-phosphate (S1P). SIP is an important signaling molecule,and serves in particular as a major regulator of vascular and immunesystems. See, e.g., Lloyd-Evans et al., Nat. Med. 14:1247-1255 (2008);Ryu et al., EMBO J. 25:5840-5851 (2006); Maceyka et al., J. Lipid Res.50:S272-S276 (2009); Peterson et al., Proc. Natl. Acad. Sci. U.S.A.105:20764-20769; and U.S. Pat. No. 6,730,480. Thus, the detection of S1Pand the sphingosine kinases is important for the understanding of thevarious systems that S1P influences. Current methods for separatingsphingosine from S1P generally use chromatographic, electrophoretic orspectrometric methods. See, e.g., Bandhuvula et al., Biochem. Biophys.Res. Commun. 380:366-370 (2009); Lee et al., Anal. Chem. 80:1620-1627(2008); Jin et al., Arch. Pharm. Res. 29:1049-1054 (2006); Jin et al.,Anal. Biochem. 380:35-40 (2008); Berdyshev et al., Anal. Biochem.339;129-136 (2005); Caligan et al., 2000, Anal. Biochem. 281:36-44(2000); and Yatomi, Biochim. Biophys. Acta 1780:606-611(2008).

Materials and Methods

The following sphingosine kinase reaction mix (50 μl) was prepared:

50 mM Tris HCl pH 8.0

150 mM NaCl

10 mM MgCl₂

1.0 mM DTT

7.5 μM Sphingosine fluoresceine

500 μM ATP

0.16 units human sphingosine kinase 1 (hSK1).

The same mix was also prepared without the enzyme, as a control and fortitration studies, as further described below.

The reaction mix was incubated 1 h at 37° C. The S1P formed by theenzyme was then precipitated in the reaction mix by the addition of 5 μlof 1 M barium acetate +300 μL of 100% ethanol, followed by 30 minincubation on ice. In some cases, 5 μl of 1 mg/ml S1P (without dye) wasadded as a carrier (normalizing reagent) to facilitate precipitation ofthe sphingosine phosphate formed. After the precipitation step, thereaction mix was vortexed and transferred to a nylon 0.22 μm filter tubeand centrifuged at 4000×g for 2 minutes. The filter tube was then washedtwice with 200 μl 70% ethanol in 1.5% Triton® X-100. The precipitate wasthen solubilized and eluted from the filter with 2×100 μl of 20 μMEDTA+100 mM Tris HCl ph 8.0+5% Triton® X-100. The eluted fluoresceinatedS1P was quantified by measuring intensity of fluorescence. Preliminarystudies established that the two wash steps were sufficient to removeessentially the entire quantity of unprecipitated sphingosinefluorescein.

Results and Discussion

The fluorescein-S1P assay described above was first performed withoutand with the S1P carrier to determine the effect of the carrier on thecompleteness of the precipitation step. Mixtures of the reaction mixwith and without hSK1were prepared, incubated, precipitated, washed, andeluted, then fluorescence emission was scanned from 510-600 nm. FIG. 2shows results where carrier was not added. The lines labeled “a” showthe results of two replications where 50 μl reaction mix with enzyme wasused without adding any reaction mix without enzyme; in “b”, 40 μlreaction mix with enzyme and 10 μl reaction mix without enzyme mix wasused; in “c”, 30 μl reaction mix with enzyme and 20 μl reaction mixwithout enzyme was used; in “d” 20 μl reaction mix with enzyme and 30 μlreaction mix without enzyme was used; in “e” 10 μl reaction mix withenzyme and 40 μl reaction mix without enzyme was used; in “f” 5 μlreaction mix with enzyme and 45 μl reaction mix without enzyme was used.As shown in FIG. 2, where 50 μl reaction mix with enzyme was usedwithout any reaction mix without enzyme (“a”), the peak of thefluorescence had an intensity of about 85,000 fluorescence counts/sec.

A graph of the peak results from these assays is shown in FIG. 3. Theassay without carrier shows linear results.

FIG. 4 shows the same study except where S1P carrier (without dye) wasadded at the precipitation step to facilitate precipitation. As showntherein, more precipitation was evident, since the peak fluorescencewhere only reaction mix with enzyme was used was about 100,000counts/second (as opposed to about 85,000 counts/sec. when no carrierwas used). Thus, in this assay, the addition of carrier resulted in anincrease in yield of precipitated fluoresceinated S1P of about 18%. Asshown in FIG. 5, the results with carrier were linear.

These assays were also utilized to demonstrate inhibition of hSK1 byknown SK1 inhibitors. The above reaction mix was prepared and the assaywas performed as described above, except 4 μM sphingosine fluoresceinewas used, with or without 100 μM of either of two SK1 inhibitorsdimethylsphingosine (DMS) and D/L sphingosine. The results are shown inFIG. 6, where the lines labeled “a” are results from two replications ofthe assay without inhibitors, the lines labeled “b” show results whenDMS was added, and the lines labeled “c” show results when D/Lsphingosine was added. As shown in FIG. 6, the inclusion of the SK1inhibitors in the assay mix caused less precipitation of thefluoresceinated product, indicating less fluoresceinated F1P resultingfrom inhibited enzyme.

EXAMPLE 2 Detection of Phosphorylated Crosstide Peptide

Example 1 showed that the assay described therein provides linear,quantitative measurements for a phosphorylated small molecule,sphingosine. That result is extended in this example by demonstratingsimilarly quantitative results for a peptide, Crosstide, with a similarassay. Crosstide is a synthetic oligopeptide (11 mer) with the sequenceGRPRTSSFAEG (SEQ ID NO:1) (using the common single letter amino acidcode), derived from glycogen synthase kinase-3 alpha (GSK-3α). It isphosphorylated at the second serine site (indicated in bold) by severalenzymes, including RAC-alpha serine/threonine-protein kinase (AKT1).

Materials and Methods

The following Crosstide reaction mix (1 ml) was prepared:

25 mM Tris HCl pH 8.0

10 mM MgCl₂

1.0 mM DTT

45.9 μM crosstide fluoresceine

500 μM ATP

0.01% Triton® X-100

10 μl AKT1 (Sigma Aldrich, St. Louis Mo.)

The same mix was also prepared without the enzyme, as a control and fortitration studies, as further described below.

The reaction mix was incubated 1 h at 37° C., then diluted 1:10 in 50 mMTris-HCl. Following this dilution, a 50 μl aliquot was taken, to which 5μl of 1 mg/ml crosstide (not fluoresceinated) was added as a carrier(normalizing reagent). Precipitation/wash buffer (200 μl), pH 5.5,consisting of 0.1 M LaNO₃, 1.0 M imidazole, 3% Triton® X-100, 0.3 M TrisHCl, and 8 ml concentrated HCl pH 5.5, was added, and the mixture wasprecipitated on ice for 1 hr.

After precipitation, the mix was transferred to a nylon 0.22 μm filtertube that was first blocked with 6% bactotryptone. The filter tube wascentrifuged for 4 minutes at 4000 rpm, and the precipitate was washedtwice with 200 μl of the precipitation/wash buffer described above. Theprecipitate was solubilized and eluted through the filter with 2×100 μlof 20 μM EDTA+50 mM Tris HCl ph 8.0+5% Triton® X-100.

Results and Discussion

FIG. 7 shows the results of this assay, where the following 6 mixturesof the reaction mix with and without the AKT1 enzyme were used: the linelabeled “a” is the result where 50 μl reaction mix with enzyme was usedwithout adding any reaction mix without enzyme; in “b”, 40 μl reactionmix with enzyme and 10 μl reaction mix without enzyme mix was used; in“c”, 30 μl reaction mix with enzyme and 20 μl reaction mix withoutenzyme was used; in “d” 20 μl reaction mix with enzyme and 30 μlreaction mix without enzyme was used; in “e” 10 μl reaction mix withenzyme and 40 μl reaction mix without enzyme was used; in “f” 0 μlreaction mix with enzyme and 50 μl reaction mix without enzyme was used.

A graph of the peak results from these assays is shown in FIG. 8. Theassay shows linear results, indicating quantitative precipitation of thefluoresceinated Crosstide.

EXAMPLE 3 Precipitation of Sulfated Peptide

Examples 1 and 2 describe assays where a dye-conjugated small molecule(Example 1) and a peptide (Example 2) is phosphorylated andquantitatively separated from the unphosphorylated substrate byprecipitation, using a metal ion. Because a phosphate functional groupis a divalent anion, those Examples do not indicate whether a compoundwith a monovalent anion functional group, such as a sulfate group, couldbe precipitated. This Example addresses that question, and establishesthat such a compound can indeed be precipitated, thus establishing thata compound with a monovalent functional group can be separated by themethods provided herein.

The following sulfated peptide (“EBJR”) was prepared by AnaSpec, Inc.(San Jose, Calif.) (using the common single letter amino acid code;5FAM=5-Carboxyfluorescein): 5FAM-GPWLEEEEEAY*GWMDF (SEQ ID NO:2). Thetyrosine residue (Y*) was sulfated.

The following quantities of EBJR was prepared in 50 μl of 50 mM Trisbuffer, pH 8.0:

Mole EBJR

5.93×10⁻¹²

2.97×10⁻¹¹

5.93×10⁻¹¹

5.93×10⁻¹⁰

3.00×10⁻⁹

6.00×10⁻⁹

To these EBJR preparations, either 1 μl of 1M phenol phosphate or 2 μl10 mM sodium sulfate was added as a carrier, along with 10 μl of 1 Mbarium acetate, 200 μl of 50 mM Tris, pH 8.0, and 5 μl of 600 mM HCl.The final pH of this solution was about 6.0. This mixture wasprecipitated on ice for 1 hr.

After precipitation, the mix was transferred to a nylon 0.22 μm filtertube that was first blocked with 6% bactotryptone. The filter tube wascentrifuged for 4 minutes at 10,000 rpm, and the precipitate (retainedon the filter) was washed with 200 μl of a wash buffer consisting of 100mM barium acetate in 50 mM Tris, pH 8.0. The precipitate was solubilizedand eluted through the filter with 2×100 μl of an elution bufferconsisting of 20 pM EDTA+50 mM Tris HCl ph 8.0+5% Triton® X-100.

FIG. 9 shows the results of the precipitation of fluoresceinated,sulfated EBJR, where the sodium sulfate carrier was used. Theprecipitation and elution of increasing concentrations of the sulfatedpeptide was linear, with minimal peptide passing through the filter asbreakthrough or in the washes, showing essentially completeprecipitation of the peptide. Results using the phenol phosphate carrierwere very similar to the results with the sodium sulfate carrier shownin FIG. 9.

These results demonstrate that compounds with monovalent anions can beprecipitated quantitatively.

In view of the above, it will be seen that he several objectives of heinvention are achieved and other advantages attained.

As various changes could be made in the above methods and compositionswithout departing from the scope of the invention, it is intended thatall matter contained in the above description and shown in theaccompanying drawings shall be interpreted as illustrative and not in alimiting sense.

All references cited in this specification are hereby incorporated byreference. The discussion of the references herein is intended merely tosummarize the assertions made by the authors and no admission is madethat any reference constitutes prior art. Applicants reserve the rightto challenge the accuracy and pertinence of the cited references,

What is claimed is:
 1. A method for detecting or quantifying an activityin a sample that causes a compound to gain a functional group, themethod comprising the steps of: (a) providing: (i) a compound which cangain a functional group, the compound comprising at least onenon-radioactive signaling moiety; (ii) a chemical source of thefunctional group which can be gained by the compound; and (iii) asample; (b) forming a mixture comprising the compound, the source of thefunctional group and the sample, and incubating the mixture underconditions suitable for the compound to gain the functional group if theactivity is present in the sample; and (c) capturing any compound thathas gained the functional group and separating said compound which hasgained the functional group from any compound which has not gained thefunctional group; and (d) detecting or quantifying the non-radioactivesignaling moiety of any captured separated compounds having thefunctional group, thereby detecting or quantifying the activity in thesample.
 2. The method of claim 1, wherein the capturing and separatingstep comprises capturing any compound that has gained the functionalgroup on a matrix.
 3. The method of claim 2, wherein the matrixcomprises a metal ion.
 4. The method of claim 3, wherein the metal ionis Fe⁺³, Ga⁺², Zn⁺³, Zn⁺², or Mn⁺².
 5. The method of claim 1, whereinthe compound comprises a macromolecule or a small biological compound.6. The method of claim 5, wherein the macromolecule or small biologicalcompound comprises a nucleic acid, a protein, a sugar, a polysaccharide,a lipid, a glycoprotein, a glycolipid, or a lipoprotein.
 7. The methodof claim 5, wherein the macromolecule or small l biological compoundcomprises an oligomer or a polymer.
 8. The method of claim 5, whereinthe oligomer or polymer comprises a nucleic acid, an abasic nucleicacid, a peptide nucleic acid, an oligo- or polypeptide, a protein, anoligosaccharide, a polysaccharide or an organic polymer.
 9. The methodof claim 1, wherein the compound comprises a protein or an oligo- orpolypeptide which is modified in post-translational modification. 10.The method of claim 9, wherein the oligo- or polypeptide is modified inpost-translational modification by a means comprising phosphorylation,acetylation, methylation, acylation, glycosylation, GPI anchor addition,hydroxylation, sulfation, disulfide bond formation, deamidation, ornitration.
 11. The method of claim 5, wherein the macromolecule or smallbiological compound comprises a lipid, a glycolipid, a lipoprotein, anapolipoprotein, a cytokine, a hormone, ceramide, a glycosylceramide, amonosaccharide ceramide, glucosylceramide, galactosylceramide, adisaccharide ceramide, lactosylceramide, a sphingosine, or asphingolipid.
 12. The method of claim 1, wherein the compound comprisesa non-biological compound.
 13. The method of claim 1, wherein thefunctional group is a phosphate, an acetyl, a methyl, an acyl, aglycosyl, a sulfate, a sulfonate, a thiol, an amine, an amide, ahydroxyl or a nitro group.
 14. The method of claim 13, wherein thefunctional group is a phosphate and the chemical source of thefunctional group is ATP.
 15. The method of claim 1, wherein thenon-radioactive signaling moiety comprises a fluorescent compound, aphosphorescent compound, a chemiluminescent compound, a chromogeniccompound, a chelating compound, an electron dense compound, a magneticcompound, an energy transfer member or pair, an intercalating compound,an antibody, an antigen, a hapten, a receptor, a hormone, a ligand or anenzyme, or any combination thereof.
 16. The method of claim 15, whereinthe capturing and separating step comprises capturing any compound thathas gained the functional group on a matrix; and the functional groupcomprises a phosphate, an acetyl, a methyl, an acyl, a glycosyl, asulfate, a sulfonate, a thiol, an amine, an amide, a hydroxyl or a nitrogroup.
 17. A method for detecting or quantifying an activity in samplewhich removes a functional group from a compound, the method comprisingthe steps of: (a) providing: (i) a compound comprising a functionalgroup and at least one non-radioactive signaling moiety; and (ii) asample; (b) forming a mixture comprising the compound and the sample andincubating the mixture under conditions suitable for the compound tolose the functional group if the activity is present in the sample; and(c) capturing and separating any compound that has the functional groupfollowing the incubation from any compound which has lost the functionalgroup; and (d) detecting or quantifying the non-radioactive signalingmoiety of any captured separated compounds having the functional group,thereby detecting or quantifying the activity in the sample.
 18. Themethod of claim 17, wherein the capturing and separating step comprisescapturing any compound that has gained the functional group on a matrix.19. The method of claim 18, wherein the matrix comprises a metal ion.20. The method of claim 93, wherein the metal ion is Fe⁺³, Ga⁺², Zn⁺³,Zn⁺², or Mn⁺².
 21. The method of claim 17, wherein the functional groupis a phosphate, an acetyl, a methyl, an acyl, a glycosyl, a sulfate, asulfonate, a thiol, an amine, an amide, a hydroxyl or a nitro group. 22.The method of claim 17, wherein the non-radioactive signaling moietycomprises a fluorescent compound, a phosphorescent compound, achemiluminescent compound, a chromogenic compound, a chelating compound,an electron dense compound, a magnetic compound, an energy transfermember or pair, an intercalating compound, an antibody, an antigen, ahapten, a receptor, a hormone, a ligand or an enzyme, or any combinationthereof.
 23. The method of claim 22, wherein the capturing andseparating step comprises capturing any compound that has gained thefunctional group on a matrix; and the functional group comprises aphosphate, an acetyl, a methyl, an acyl, a glycosyl, a sulfate, asulfonate, a thiol, an amide, a hydroxyl or a nitro,